The present invention relates to biologically degradable polypeptides which possess active halogen atoms which are covalently bound to side chains; to such polypeptides which possess recognition molecules and/or water-soluble groups which are bound in the side chains; and also to processes for their preparation by reacting polyamides which contain functional groups with halomethylcarbonylating agents or reacting the polypeptides which contain active halogen atoms with this group-containing recognition molecules or water-soluble groups; and to the use of these polymers in therapeutic compositions or in the active layer of diagnostic agents.
Receptor-ligand interactions are of great importance in intracellular communication and in intercellular recognition processes. The initiation of diseases, for example bacterial diseases [J. C. Paulson, The receptors, Vol. II, Ed.: P. M. Conn, Academic Press, 1985, 131], viral Infections [Strxc3x6mberg et al. EMBO J. 1990, (9), 2001] and inflammatory diseases [Dasgupta, F., Rao, B. N. N., Exp. Opin. Invest. Drugs 3:709-724 (1994)] takes place by way of ligand-receptor interactions. In specified examples, the ligands are oligosaccharides. It is not possible to use free oligosaccharides, which are to bind to receptors in place of natural ligands, for the therapy of these diseases because of the very high quantities of oligosaccharides which have to be administered since the affinity between the receptors and the ligands is too low.
It is known that an increased interaction between receptor and ligand is achieved by coupling several ligands on a surface. The example of the viral protein haemagglutinine, binding to neuramic acid on the cell surface and their interaction has been used to show how this polyvalent effect obtained by using a polymer affects such an interaction [A. Spaltenstein et al. J. Am. Chem. Soc. 1991 (113) 686].
Polymeric compounds which present ligands in multivalent form can lead, in ligand-receptor recognition processes, to increased interactions with receptors and be used therapeutically, for example as receptor blockers [W. J. Lees et.al., J. Med. Chem. 1994, 37, 3419; EP 601417 A2] or multivalent enzyme inhibitors [WO 90/02558].
Other applications have also been reported for polymers which are functionalized by defined quantities of active molecules. For targeting active substances, use can be made of more complex polymers which, in addition to active compounds, also carry substances which are selectively recognized by particular cell-surface receptors so that these active compounds are preferentially transported to these types of cells [J. Kopecjek, R. Duncan, J. Controlled Release 1987, 6, 315].
In addition, active compounds which are coupled covalently to polymers by way of labile bonds can be selectively released in the organism at sights where these bonds are cleaved by particular physiological conditions [B. Zorc et. al., Acta Pharm. 1994, 44,103; B.Zorc et. al., Int. J. Pharmaceutics 1993, 99, 135; G. Giammona et. al., Eur. J. Pharm. Biopharm. 1992, 38, 159; G. Giammona et. al., Eur. J. Pharm Biopharm. 1992, 38, 159; G. Giammona et. al. Int J. Pharmaceutics 1989, 57, 55].
Polymers which are functionalized by receptor molecules or ligands can be widely used in diagnostics. Immobilized marker molecules, for example biotin, can be used when carrying out biological tests [N. E. Nifant""ev et. al., in xe2x80x9cSynthetic Oligosaccharidesxe2x80x9d, Ed.: P. Kovac, ACS Symposium Series 560, Washington, D.C. 1994)]. Compounds of this nature can be present as recognition elements in the active layers of sensors [Janata, Principles of Chemical Sensors, Plenum Press, New York 1989].
Immobilized active molecules can also be antigens which can be employed as vaccines [Conjugate Vaccines, Eds.: J. M. Cruse, R. E. Lewis, Jr; Karger, Basel 1989; Towards Better Carbohydrate Vaccines, Eds.: R. Bell, G. Torrigiani, John Wiley and Sons, Chichester 1987].
EP 0601417 A2 already discloses a mixture in which physiologically tolerated and physiologically degradable polymer-based carbohydrate receptor blockers are prepared. These blockers consist of a carbohydrate moiety, a bifunctional spacer, a hydrophilic polymer and an effect enhancer. However, it is not possible to incorporate all or some of the carbohydrate ligands in a specific manner or even to incorporate different ligands in a specific manner.
For the purpose of constructing complex polymers in a controlled manner, small quantities of an active substance can be coupled to a preformed polymer which carries an excess of activated side chains. This principle was exploited for synthesizing polymeric, water-soluble neoglycoconjugates which are based on nitrogen-substituted polyacrylamides [WO 94/11005-A1; N. V. Bovin et. al., Glycoconjugate J. 1993, 10, 142; N. E. Nifant""ev et al., in xe2x80x9cSynthetic Oligosaccharidesxe2x80x9d, Ed.: P. Kovac, ACS Symposium Series 560, Washington, D.C. 1994]. However, the scope for using such compounds is limited by the fact that the polymer backbone is not necessarily biodegradable.
It has now been found, surprisingly, that biodegradable polymers which possess side chains which are functionalized identically or differently can be specifically obtained with a precisely defined, predictable composition when polypeptides which possess activated halogen atoms in the side chains are used as the starting material and are reacted with substituents which contain a mercapto group. In addition, these polypeptides can be prepared in a simple manner and in high yields and purities. Water-soluble, gelatinous or water-insoluble potypeptides can be obtained depending on the hydrophilicity of the substituents. It is particularly worth mentioning that precisely defined active compound densities can be set and it is possible, concomitently to carry out further modification which is controlled in accordance with the desired application. The polypeptides which contain activated halogen atoms are also surprisingly easy to obtain, very stable and readily soluble in various organic solvents.
The invention relates to polypeptides which possess identical or different structural elements of the formula (I), 
in which
A is a trivalent, aliphatic hydrocarbon radical having from 1 to 12 C atoms which is unsubstituted or substituted by one or more substituents selected from the group consisting of C1-C4alkyl, C1-C4alkoxy, C1-C4alkylthio, benzyl and benzyloxy,
R1 is a direct bond or C1-C6alkylene,
X1 is xe2x80x94C(O)Oxe2x80x94,xe2x80x94C(O)NRxe2x80x94, xe2x80x94NRxe2x80x94, xe2x80x94Sxe2x80x94 or xe2x80x94Oxe2x80x94;
R2 is a bivalent bridging group,
X2 is O or NR, or R2 and X2 are together a direct bond,
X3 is a halogen, and
R is H or C1-C6alkyl;
with the proviso that X1 is not xe2x80x94NRxe2x80x94, xe2x80x94Sxe2x80x94 or xe2x80x94Oxe2x80x94 when R1 is a direct bond.
As halogen, X3 is preferably Cl, Br or I, and particularly preferably Cl or Br.
The trivalent radical A preferably contains from 1 to 8, more preferably from 1 to 6, particularly preferably from 1 to 4 and, in particular, 1 or 2, C atoms. Examples are 1,1,6-, 1,2,6-, 1,3,6-, 1,4,6-, 1,5,6- or 1,6,6-hexanetriyl, 1,1,5-, 1,2,5-, 1,3,5-, 1,4,5- or 1,5,5-pentanetriyl, 1,1,4-, 1,2,4-, 1,3,4- or 1,4,4-butanetriyl, 1,1,3-, 1,2,3- or 1,3,3-propanetriyl, 1,1,2- or 1,2,2-ethanetriyl and methanetriyl. Methanetriyl and 1,1,2- and 1,2,2-ethanetriyl are particularly preferred.
As alkylene, R1 preferably contains from 1 to 6 C atoms, more preferably 1-4 C atoms, which can be linear or branched. Examples are 1,6-, 1,5-, 1,4-, 1,3-, 1,2-, 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, 3,5- or 3,6-hexylene, 1,5-, 1,4-, 1,3-, 1,2-, 2,3-, 2,4-, 2,5-, 3,4- or 3,5-pentylene, 1,4-, 1,3-, 1,2-, 2,3-, 2,4- or 3,4-butylene, 1,3-, 1,2- or 2,3- propylene, 1,2-ethylene and methylene. R1 is particularly preferably linear and is especially preferably methylene, ethylene, 1,3 propylene or 1,4-butylene.
Within the scope of the present invention, X1 is preferably NR or xe2x80x94C(O)xe2x80x94NRxe2x80x94, with R particularly preferably being H.
The bridging group can contain from 1 to 30, more preferably from 1 to 20, particularly preferably from 1 to 12, and especially preferably from 1 to 8, atoms selected from the group C, O, S, P and N. The bivalent bridging group R2 can be C2-C8hydrocarbon radicals which are interrupted by an xe2x80x94NHxe2x80x94C(O)xe2x80x94 group or be C2-C8hydrocarbon radicals which are covalently bound by way of an xe2x80x94NHxe2x80x94 group. The bridging group is preferably hydrocarbon radicals which can be interrupted by one or more heteroatoms from the group O, S, P and N, and/or by xe2x80x94C(O)xe2x80x94Oxe2x80x94, C(O)xe2x80x94Nxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94Nxe2x80x94C(O)xe2x80x94Nxe2x80x94 and xe2x80x94C(O)xe2x80x94Oxe2x80x94 groups.
The bridging group can, for example, conform to the formula (VI),
xe2x80x94R4xe2x80x94X5xe2x80x94(R5)rxe2x80x94(X4)sxe2x80x94R6xe2x80x94xe2x80x83xe2x80x83(VI),
in which
X5 and X4 are, independently of each other, a direct bond, or X5 and X4 are, independently of each other, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NR7xe2x80x94,xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94SO2xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94SO2xe2x80x94, xe2x80x94Oxe2x80x94SO2xe2x80x94O, xe2x80x94NR7xe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94NR7xe2x80x94, xe2x80x94NR7xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94NR7xe2x80x94, xe2x80x94NR7xe2x80x94C(O)xe2x80x94NR7xe2x80x94, xe2x80x94NR7SO2xe2x80x94, xe2x80x94SO2xe2x80x94NR7xe2x80x94, xe2x80x94NR7xe2x80x94SO2xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94SO2NR7xe2x80x94, xe2x80x94NR7SO2xe2x80x94NR7xe2x80x94 or 
xe2x80x83R5 is a bivalent bridging group,
R7 is H, C1-C12alkyl, C5- or C6cycloalkyl, C5- or C6cycloalkylmethyl or -ethyl, phenyl, benzyl or 1-phenyleth-2-yl,
R4 and R6 are, independently of each other, a direct bond, C1-C18alkylene, C5- or C6-cycloalkylene, C6-C10arylene or C7-C12aralkylene,
r is the numbers 0 or 1 and s is the numbers 0 or 1, and s is 0 when r is 0,
X8 is OH, its salts, for example alkali metal, alkaline earth metal or ammonium salts (Na, K, Ca, Mg), or NR8R9, and X9 and X10 are, independently of each other, O or NR8, with R8 and R9 being, independently of each other, H, C1-C12alkyl, C5- or C6cycloalkyl, C5- or C6-cycloalkylmethyl or -ethyl, phenyl, benzyl or 1-phenyleth-2-yl.
In the meaning of alkyl, R7 preferably contains from 1 to 6, and particularly preferably from 1 to 4, C atoms. Some examples are methyl, ethyl, n- or i-propyl, butyl, hexyl and octyl. In the meaning of cycloalkyl, R7 is preferably cyclohexyl, and cyclohexylmethyl in the meaning of cycloalkylmethyl. In a preferred embodiment, R7 is H or C1-C4alkyl.
The bivalent bridging group is preferably a hydrocarbon radical which preferably contains from 1 to 30, more preferably from 1 to 18, particularly preferably from 1 to 12 and especially preferably from 1 to 8, C atoms and which is unsubstituted or substituted once or more than once by C1-C4alkyl, C1-C4alkoxy or xe2x95x90O. The hydrocarbon radical can also be interrupted once or more than once by heteroatoms selected from the group xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94 and xe2x80x94NR2xe2x80x94, with R2 preferably being H or C1-C4alkyl.
The bivalent bridging group can, for example, be C1-C20xe2x80x94, preferably C2-C12xe2x80x94, alkylene which can be linear or branched. Some examples are methylene, ethylene, 1,2- or 1,3-propylene, 1,2-, 1,3- or 1,4-butylene, pentylene, hexylene, octylene, dodecylene, tetradecylene, hexadecylene and octadecyiene.
The bivalent bridging group can, for example, be polyoxaalkylene having from 2 to 12, preferably from 2 to 6, and particularly preferably from 2 to 4, oxaalkylene units and from 2 to 4, preferably 2 or 3, C atoms in the alkylene. Particularly preferably, the bridging group is polyoxaethylene and polyoxapropylene having, for example, from 2 to 6 oxaalkylene units in the bridging group.
The bivalent bridging group can, for example, be C5-C12xe2x80x94, preferably C5-C8xe2x80x94, and particularly preferably C5- or C6cycloalkylene, for example cyclopentylene, cyclohexylene, cyclooctylene or cyclododecylene.
The bivalent bridging group can, for example, be C5-C12xe2x80x94, preferably C5-C8xe2x80x94, and particularly preferably C5- or C6cycloalkyl-C1-C12xe2x80x94 and preferably xe2x80x94C1-C4-alkylene. Some examples are cyclopentylene-CnH2nxe2x80x94 and cyclohexylene-CnH2nxe2x80x94, in which n is a number from 1 to 4. Cyclohexylene-CH2xe2x80x94 is particularly preferred.
The bivalent bridging group can, for example, be C6-C14arylene and preferably C6-C10arylene, for example naphthylene or, more preferably, phenylene.
The bivalent bridging group can, for example, be C7-C20aralkylene and preferably C7-C12aralkylene. Greater preference is given to arylene-CnH2nxe2x80x94, in which arylene is naphthylene and, particularly, phenylene, and n is a number from 1 to 4. Examples are benzylene and phenylethylene.
The bivalent bridging group can, for example, be arylene-(CnH2nxe2x80x94)2xe2x80x94, in which arylene is preferably naphthylene and especially phenylene, and n is a number from 1 to 4. Examples are xylylene and phenylene(CH2CH2)2xe2x80x94.
The hydrophilicity of the polymers can be adjusted by the choice of the bivalent bridging groups. In a preferred embodiment, the bridging group is an oxaalkylene or a polyoxaalkylene preferably having from 2 to 4 C atoms, particularly preferably 2 or 3 C atoms, in the alkylene and from 2 to 20, preferably from 2 to 10, alkylene units. Particular preference is given to oxaethylene and polyoxaethylene having from 2 to 20, preferably from 2 to 10, oxaethylene units.
Within the scope of the present invention, X2 is preferably O.
In a preferred embodiment, R2xe2x80x94X2 is a direct bond.
In its meaning as alkyl, R preferably contains 1-4 C atoms. Examples are methyl, ethyl and the isomers of propyl and butyl. R is preferably H.
A preferred subgroup of the novel polypeptides contains structural elements of the formula Ia, 
in which
R01 is linear C2-C6xe2x80x94, and particularly preferably C2-C4alkylene,
X01 is the group xe2x80x94NHxe2x80x94, and
X03 is Cl or Br.
The novel polypeptides can be homopolymers, copolymers having at least two structural elements of the formula I, or copolymers additionally having comonomer units of other aminocarboxylic acids. The copolymers can be block polymers or statistical polymers.
In one embodiment of the subject-matter of the invention, the polypeptides having the structural elements of the formula (I) additionally contain at least one structural element of the formula (II), or additionally contain at least two different structural elements of the formula II, 
in which
Z1 is H or My 
A, R1, X1, R2 and X2 are as defined above and y is 1 and M is a monovalent metal or y is xc2xd and M is a divalent metal; including their physiologically tolerated salts.
In another embodiment of the subject-matter of the invention, the polypeptides having the structural elements of the formula (I) and, if desired, structural elements of the formula II additionally contain at least one structural element of the formula (IV), or at least two different structural elements of the formula IV, 
in which
A1 is a divalent aliphatic hydrocarbon radical having from 1 to 12 C atoms which is unsubstituted or substituted by one or more substituents selected from the group consisting of C1-C4aminoalkyl, C1-C4hydroxyalkyl, HOOCxe2x80x94C1-C4alkyl, C1-C4alkyl, C1-C4alkoxy, C1-C4alkylthio, benzyl or benzyloxy.
For the purposes of the present invention, a metal is to be understood as meaning an alkali metal [for example lithium (Li), sodium (Na), potassium (K), rubidium (Rb) and caesium (Cs)], an alkaline earth metal (for example magnesium (Mg), calcium (Ca) and strontium (Sr)) or manganese (Mn), iron (Fe), zinc (Zn) or silver (Ag). Physiologically tolerated salts are to be understood as meaning, in particular, the alkali metal and alkaline earth metal salts, for example sodium, potassium, magnesium and calcium salts. Sodium and potassium ions and their salts are preferred.
As alkyl, the divalent radical A1 can be linear or branched and preferably contains from 1 to 8, more preferably from 1 to 6, particularly preferably from 1 to 4, and in particular 1 or 2, C atoms. Examples are 1,6-, 1,5-, 1,4-, 1,3-, 1,2-, 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, 3,5- or 3,6-hexylene, 1,5-, 1,4-, 1,3-, 1,2-, 2,3-, 2,4-, 2,5-, 3,4- or 3,5-pentylene, 1,4-, 1,3-, 1,2-, 2,3-, 2,4- or 3,4-butylene, 1,3-, 1,2- or 2,3- propylene, 1,2-ethylene and methylene.
A1 is particularly preferably linear and is in particular C1-C4alkylene.
Those substituents for A1 are preferred which occur in natural amino acids, i.e. amino-, hydroxy- and carboxyalkyl, for example.
In a preferred embodiment, the bivalent radicals A1 are derived from natural amino acids with the exception of unprotected cysteine.
In the novel polypeptide, the content of the structural elements I, II and IV can, for example, be from 100 to 0.1 mol %, preferably from 100 to 5.0 mol %, more preferably from 100 to 10 mol %, particularly preferably from 100 to 30 mol %, and in particular from 100 to 50 mol %, in the case of structural elements of the formula (I), from 0 to 99.9 mol %, preferably from 0 to 95 mol %, more preferably from 0 to 90 mol %, particularly preferably from 0 to 70 mol %, and in particular from 0 to 50 mol %, in the case of structural elements of the formula (II), and from 0 to 99.9 mol %, preferably from 0 to 95 mol %, more preferably from 0 to 90 mol %, particularly preferably 0-70 mol %, and in particular from 0 to 50 mol %, in the case of structural elements of the formula (IV).
The content of structural elements of the formula IV which are derived from natural amino acids, with the exception of unprotected cysteine, can, for example, be from 0 to 99.9 mol %, preferably from 0 to 95 mol %, more preferably from 0 to 90 mol %, particularly preferably from 0 to 70 mol %, and in particular from 0 to 50 mol %.
The values in mol % always add up to 100%.
The mean molar mass of the polymer is, for example, at least 2 kDa, preferably 10 kDa, more preferably 20 kDa, particularly preferably 30 kDa, and can be up to 500 kDa, more preferably up to 100 kDa and particularly preferably up to 70 kDa.
The novel polymers may be obtained in a simple manner and, surprisingly, in very high yields and at high purities by reacting polypeptides which contain appropriate functional groups with ester-forming or amide-forming derivatives of haloacetic acids.
The invention furthermore relates to a process for preparing polymers having identical or different structural elements of the formula (I) and, if desired, structural elements of the formulae II and IV, 
in which A, R1, X1, R2, X2 and X3 are as defined above, which comprises reacting polymers having identical or different structural elements of the formula (II), and, if desired, identical or different structural elements of the formula IV, 
in which
Z1, A, R1, X1, R2 and X2 are as defined above, with an amide-forming or ester-forming derivative of a monohaloacetic acid in the presence of an acid-capturing agent.
The amide-forming or ester-forming derivative can, for example, be an acid anhydride or acid halide, for example those of the formulae IIIa or IIIb, 
in which X3 is as defined above and X4 is preferably chlorine or bromine; or the derivatives can be activated esters of the formula IIIc, 
in which X3 is as defined above and Y is, for example, pentafluorophenyl-, p-nitrophenyl-, 
in which R07 and R08 are cyclohexyl or Isopropyl.
Chloroacetic anhydride is particularly preferably used.
Examples of suitable acid-capturing agents are organic nitrogen bases, in particular tertiary amines having, for example, from 3 to 30, preferably from 3 to 20, and particularly preferably from 3 to 12 C atoms. The tertiary amine is preferably a sterically hindered, non-nucleophilic tertiary amine, preferably a cyclic, sterically hindered, non-nucleophilic tertiary amine. The cyclic amines can be of an aliphatic or aromatic nature and they are substituted in one or both positions which are ortho to the N atom, for example by alkyl groups which contain from 1 to 4 C atoms, for example methyl or ethyl. Aromatic amines which are substituted in both ortho positions are preferred. A particularly preferred example is 2,6-lutidine.
The acid-capturing agent can be present in the reaction mixture stoichiometrically or as a cosolvent.
The reaction temperature is advantageously from xe2x88x9240 to +100xc2x0 C., preferably from xe2x88x9220 to +70xc2x0 C., particularly preferably from xe2x88x9210 to +50xc2x0 C.
The reaction advantageously takes place in the presence of a polar or apolar, aprotic solvent. Preference is given to nitrogen-dialkylated carboximides and lactams, sulfoxide and sulfones, for example dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, and tetramethylene sulfone.
The novel polypeptides are outstandingly suitable for preparing, in a specific manner and at exceptionally high purities, polypeptides which contain recognition molecules and which have precisely defined contents, with it additionally being possible to set desired properties such as water-solubility, swellability or water-insolubility in a precise manner. The novel polymers are biologically degradable and therefore particularly suitable for applications in the physiological sphere, with it being possible to match the polymer properties precisely to the existing requirements. The ease of preparation by means of replacing the halogen group X3 with compounds containing thiol groups is particularly worth mentioning.
The invention furthermore relates to polypeptides having identical or different structural elements of the formula (V) 
in which A, R1, X1, R2 and X2 are as defined above, and R3 is a biological active group which is covalently bound to the S atom either directly or by way of a bridging group Y.
Biological active group means that there is a receptor function or ligand function or that the swelling or solubility properties in water are affected or that a receptor function or ligand function is combined with the alteration in the swellability or solubility properties. The biological active group consequently has a ligand function, or the active group additionally or only has a swellability or solubility-altering function.
Direct linkage means that the biological active group R3 is bound to a side chain of the polymer by way of an S atom. Bound by way of a bridging group Y1 means that the S atom is bound to a bridging group Y1 and to a side chain of the polymer, with it being possible for the bridging group Y1 to be bound to the biological active group R3 either directly or by way of a functional group.
The bridging group Y1 for R3 can, independently, have the same meanings as the bridging group R2, including the preferences. Preferred examples are C1-C18alkylene which is uninterrupted or is interrupted by one or more, preferably one to three, xe2x80x94NHxe2x80x94COxe2x80x94 groups, and, more preferably, C1-C2alkylene, and, particularly preferably, C3-C8alkylene which is not interrupted or is interrupted by an xe2x80x94NHxe2x80x94COxe2x80x94 group. An advantageous bridging group Y1 is oligo- or poly-ethylene glycol.
R4 and R6 are preferably, independently of each other, C1-C12alkylene which is unsubstituted or substituted by one or more substituents selected from the group consisting of OH, amino, C1-C4 alkyl, C1-C4 alkoxy or C1-C8 acylamino. R4 and R6 are more preferably, independent of each other, C1-C6 alkylene which is unsubstituted or substituted by one or more substituents selected from the group consisting of OH, amino, C1-C4 alkyl, C1-C4 alkoxy or C1-C8 acylamino. Particularly preferred examples are methylene, ethylene, 1,2- and 1,3-propylene and 1,2-, 1,3- and 1,4-butylene. As arylene, R4 and R6 are preferably phenylene and as aralkylene, R4 and R6 are preferably benzylene.
The novel polypeptides can be homopolymers or copolymers having different biological active groups R3.
In another embodiment, the novel polypeptides can be copolymers which are composed of at least one structural element of the formula (V) and at least one further structural element of the formulae (I), (II) and (IV). The bivalent peptide residues of the natural amino acids, with the exception of unprotected cysteine, are particularly suitable as structural elements of the formula IV.
In a further embodiment, the novel polypeptides contain at least one structural element of the formula (Va) 
in which A, R1, R2, and X2 are as defined above, and R03 is a radical of the formula (IX),
xe2x80x94C1-C12alkylene(X6)xe2x80x94Oxe2x80x94Hxe2x80x83xe2x80x83(IX)
in which X6 is CO, SO2, Pxe2x95x90O, NR11SO2 or Oxe2x80x94Pxe2x95x90O, in which R11 is C1-C6 alkyl or H, or their salts, for example alkali metal, alkaline earth metal or ammonium salts (Na, K, Ca or Mg), or R03 is a polyhydroxyalkyl or cycloalkyl radical having preferably from 2 to 12 and particularly preferably from 2 to 6 C atoms and also from 1 to 6 and preferably from 2 to 5 hydroxyl groups, or a copoymer having structural elements of the formula (Va) and at least one of the structural elements of the formulae I, II and IV. The alkylene group in formula (IX) is preferably C1-C6alkylene, particularly preferably C1-C4alkylene.
A preferred subgroup of the novel polypeptides are copolymers having structural elements of the formulae (V) and (Va). The structural elements of the formula V are preferably present in a quantity of from 0.1 to 50, more preferably of from 0.5 to 40, and particularly preferably of from 1 to 20 mol %, and the structural elements of the formula Va are preferably present in a quantity of from 99.9 to 50, more preferably of from 99.5 to 60, and particularly preferably of from 99 to 80 mol %.
The embodiments and preferences which were previously presented for the structural elements of the formulae I, II and IV also apply to the polypeptides having structural elements of the formula V.
The content of the structural elements in the polypeptide can, for example, be from 100 to 0.1 mol %, preferably from 100 to 5.0 mol %, more preferably from 100 to 10 mol %, particularly preferably from 100 to 30 mol %, and in particular from 100 to 50 mol % for structural elements of the formula (V), from 0 to 99.9 mol %, preferably from 0 to 95 mol %, more preferably from 0 to 90 mol %, particularly preferably from 0 to 70 mol % and in particular from 0 to 50 mol % for structural elements of the formula (I), from 0 to 99.9 mol %, preferably from 0 to 95 mol %, more preferably from 0 to 90 mol %, particularly preferably 0-70 mol %, and in particular from 0 to 50 mol %, for structural elements of the formula (II), from 0 to 99.9 mol %, preferably from 0 to 95 mol %, more preferably from 0 to 90 mol %, particularly preferably from 0 to 70 mol %, and in particular from 0 to 50 mol % for structural elements of the formula (IV). The content of natural amino acid residues of the formula (IV), with the exception of unprotected cysteine, can be from 0 to 99.9 mol %, preferably from 0 to 95 mol %, more preferably from 0 to 90 mol %, particularly preferably from 0 to 70 mol %, and in particular from 0 to 50 mol %.
The values in mol % always add up to 100%.
The molecular weight of the polypeptide can, for example, be at least 2 kDa, preferably at least 20 kDa, and it can be up to 500 kDa, more preferably up to 300 kDa and particularly preferably up to 250 kDa.
As a biological active group, R3 is preferably a monovalent or oligovalent ligand which is recognized and bound by a receptor molecule. Preferred ligands are biologically active molecules, for example pharmaceuticals, particularly preferably alkaloids which are highly active pharmacologically; preference is given to biologically active molecules such as carbohydrates, particularly preferably oligosaccharides and polysaccharides, preferably structures or corresponding mimetics as occur on glycoproteins and glycolipids; preference is also given to biologically active molecules such as vitamins, particularly preferably biotin and biotin analogues; preference is also given to biologically active molecules such as peptides and proteins, with active compounds being particularly preferred which have an oligopeptide or polypeptide structure or relatively low molecular weight, in particular active compounds having an oligopeptide or polypeptide structure of relatively low molecular weight which possess a hormonal (peptide hormones), antibiotic (peptide antibiotics) or toxic (peptide toxins) effect; preference is also give to conjugated proteins such as glycoproteins and glycolipids; preference is also given to biologically active molecules such as lipids; preference is also given to biologically active molecules such as terpenes, particularly preferably bitter substances, pheromones, carotinoids, insecticides, cytostatications and antibiotics; preferred ligands are biologically active molecules such as oligonucleotides, particularly preferably RNA and DNA; preference is also given to biologically active molecules such as antigens, more preferably their antigenic determinants and particularly preferably the hapten itself, in particular viral antigens, bacterial antigens, antigens of parasites, viral surface proteins, bacterial surface proteins, surface proteins of parasites, insecticides and toxins; preference is also given to biologically active molecules such as antibodies, particularly preferably immunoglobulins G, M, A and E, more preferably antibodies which are directed against viral antigens, bacterial antigens, antigens of parasites, tumour antigens, viral surface proteins, bacterial surface proteins, oligosaccharide sequences of crosslinked polysaccharides as occur on bacterial cell walls, surface proteins of parasites, insecticides and toxins.
For example, R3 is particularly preferably a monosaccharide, oligosaccharide or polysaccharide residue, for example a carbohydrate residue of the formula Cn(H2O)n and also polyhydroxy aldehydes, polyhydroxy ketones, polyhydroxy acids and polyhydroxy amines which are derived therefrom.
The carbohydrate residue can consist of from 1 to 20 naturally occurring sugar monomers and also modified sugar monomers. Use is preferably made of from 1 to 15, and particularly preferably of from 1 to 10, naturally occurring sugar monomers. The skilled person is familiar with naturally occurring sugar monomers from the standard works of organic chemistry or biochemistry, for example from Beyer/Walter, xe2x80x9cLehrbuch der organischen Chemie (Textbook of organic chemistry)xe2x80x9d, S. Hirzel Verlag Stuttgart, 21st edition, pp. 425ff.; Albert Lehninger, xe2x80x9cBiochemie (Biochemistry)xe2x80x9d, 2nd edition, pp. 201ff., VCH-Verlagsgesellschaft Weinheim, Germany, or Lubert Streyer, xe2x80x9cBiochemie (Biochemistry)xe2x80x9d, Spektrum-der-Wissenschaft Verlagsgesellschaft GmbH, Heidelberg, Germany, 1st edition, pp. 345 ff., and J. F. Kennedy, ed., xe2x80x9cCarbohydrate Chemistryxe2x80x9d, Clarendon Press, Oxford, 1988, pp. 4 ff.).
Examples of sugar monomers are selected from the group consisting of D- and L-aldo-pyranoses and D- and L-aldofuranoses, for example glyceraldehyde, erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose and talose, from the group consisting of D- and L-ketopyranoses and D- and L-ketofuranoses, for example dihydroxyacetone, erythrulose, ribulose, xylulose, psicose, fructose, sorbose and tagatose, and also from the group consisting of D- and L-diketopyranoses, for example pentodiulose and hexodiulose.
The term sugar monomers also includes those sugar monomers which are modifications of the examples listed. The skilled person understands these modifications to include, for example, protected, partially protected or unprotected deoxysugars of the D- and L-configurations, preferably 2-, 3-, 4-, 5- and 6-deoxyaldoses, such as fucose, rhamnose and digitoxose, 1,2-dideoxyaldoses, such as glucal, galactal and fucal, and 1-, 3-, 4-, 5- and 6-deoxyketoses, 2-, 3-, 4-, 5- and 6-deoxyaminosugars of the D and L configurations, such as glucosamine, mannosamine, galactosamine and fucosamine, and deoxyacylamino-sugars, such as N-acylglucosamine, N-acylmannosamine, N-acylgalactosamine and N-acyl-fucosamine, preferably their C1-C4alkyl esters.
In addition, these modifications are understood to mean aldonic, aldaric and uronic acids, such as gluconic acid or glucuronic acid, and also ascorbic acid, amino acid-carrying sugar monomers and those which carry lipid, phosphatidyl or polyol residues.
Modified sugar monomers are also understood to mean those having a carbon chain which is longer than 6 C atoms, such as heptoses, octoses, nonoses, heptuloses, octuloses and nonuloses, and also their representatives which are substituted in accordance with the above-listed criteria, such as ketodeoxyoctanic acid, ketodeoxynonanic acid, N-acyl-neuraminic acids and N-acylmuraminic acids.
Within the scope of the present invention, dimeric and trimeric sugars are understood to mean those which are assembled from two or three of the abovementioned, identical or different, monomers. The linkage is preferably xcex1-O-glycosidic or xcex2-O-glycosidic, with, however, S-, N- and C-glycosidic linkages also coming into consideration. All the C atoms of the one partner of a bond come into consideration. Examples are, in particular, (1xe2x86x922)-, (1xe2x86x923)-, (1xe2x86x924)-, (1xe2x86x925)-, (1xe2x86x926)-, (2xe2x86x923)- and (2xe2x86x926)-glycosidic linkages. Examples sugars are selected from the group consisting of trehalose, sophorose, kojibiose, laminaribiose, maltose, cellobiose, isomaltose, gentibiose, sucrose and lactose, and their derivatives. Examples of trimeric sugars are raffinose and melezitose.
Examples of particularly preferred embodiments of the carbohydrate moiety are sialyl-Lewis X or sialyl-Lewis A and also analogues of sialyl-Lewis X or sialyl-Lewis A.
R3 is preferably a C1-C8alkyl which is substituted by xe2x80x94CO2H or xe2x80x94SO3H, or their alkali metal or alkaline earth metal salts. Examples are xe2x80x94CH2CH2SO3Na and xe2x80x94CH2xe2x80x94COONa.
R3 is preferably an oligonucleotide. The oligonucleotide can be partially or completely composed of natural DNA building blocks or unnatural synthetic nucleotides. Synthetic building blocks comprise the modifications of natural building blocks in the nucleic acid base, the furanose ring and/or the bridging groups of the oligonucleotides. In general, synthetic building blocks are employed in order to enhance the complex formation in duplex structures and/or increase the stability of the oligonucleotides towards the degradation which is caused, for example, by nucleases. Within the field of antisense technology, modified nucleosides for synthesizing or modifying complementary oligonucleotides have become known in large numbers and are not therefore discussed here in more detail (see, for example, E. Uhlmann et al., Chemical Reviews, Volume 90, Number 4, pages 543 to 584 (1990)).
Suitable modifications are modifications in the nucleic base part (for example substitutions or omission of substituents), in the nucleotide bridging group (for example modification of the phosphoric ester group or its replacement with other bridging groups) and in the furanose ring (for example substitutions on the 2xe2x80x2-hydroxyl groups, replacement of the furanose O atom, replacement of the furanose ring with monocarbocyclic or bicarbocyclic rings and replacement of the furanose ring with open-chain structures).
The oligonucleotides can, for example, contain from 5 to 100, preferably from 5 to 50, particularly preferably from 8 to 30, and very particularly from 10 to 25, building blocks.
The oligonucleotides are preferably composed of nucleosides from the purine and pyrimidine series. They are particularly preferably composed of 2xe2x80x2-deoxy-2-aminoadenosine, 2xe2x80x2-deoxy-5-methylcytosine, 2xe2x80x2-deoxyadenosine, 2xe2x80x2-deoxycytidine, 2xe2x80x2-desoxycytidine, 2xe2x80x2-deoxy-guanosine and 2xe2x80x2-thymidine. Very particular preference is given to 2xe2x80x2-deoxyadenosine (A), 2xe2x80x2-deoxycytidine (C), 2xe2x80x2-deoxyguanosine (G) and 2xe2x80x2-thymidine (T). Modified building blocks are preferably derived from natural nucleosides of the purine and pyrimidine series, particularly preferably from adenosine, cytidine, guanosine, 2-aminoadenosine, 5-methylcytosine, thymidine and the previously mentioned deoxy derivatives. The nucleosides can be 2xe2x80x2-modified ribonucleosides.
In a very particularly preferred embodiment of the invention, the oligonucleotide is composed of natural deoxynucleosides, particularly preferably from the group 2xe2x80x2-deoxyadenosine (A), 2xe2x80x2-deoxycytidine (C), 2xe2x80x2-deoxyguanosine (G), and 2xe2x80x2-thymidine (T), or of complementary, unnatural synthetic building blocks. Within the scope of the invention, those modified nucleosides are particularly preferred which increase the stability of the oligonucleotide towards nucleases.
The oligonucleotide can also consist of sequences of peptide nucleic acid (PNA). The same preferences as for the oligonucleotides apply for constructing the PNA sequence. Examples of PNA""s are to be found in Science, Volume 254, pages 1497 to 1500.
The oligonucleotide can be partially or completely composed of natural DNA building blocks which are complementary to the target RNA or target DNA or be completely composed of unnatural synthetic nucleotides which are likewise complementary to the target RNA or target DNA, with partially meaning that natural DNA building blocks which are complementary to the target RNA are replaced in the oligonucleotide sequence with unnatural synthetic nucleotides which are likewise complementary.
Within the scope of the present invention, target RNA means that an RNA sequence must be present in the target. Accordingly, polyribonucleic acids (RNA) can be present. This RNA is preferably mRNA (messenger RNA), pre-mRNA (precursor mRNA), tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA) and viral RNA. However, mixed sequences of RNA and polydeoxyribonucleic acids (DNA) can also be present, for example the RNA-DNA chimeras (Okazaki fragments). The RNA possesses sufficient building blocks to allow a complex (double strand) to be formed with the oligonucleotide. Within the scope of the present invention, target DNA means that a complementary DNA sequence must be present in the target. The DNA possesses sufficient building blocks to allow a complex (double strand) to be formed with the oligonucleotide.
The choice and the order of the building blocks in a sequence of oligonucleotide is determined by the necessary formation of a duplex with a target RNA or target DNA.
The number of the building blocks in the oligonucleotide is calculated to ensure that hybridization takes place with the target RNA or target DNA. The regions (pairing nucleotide building blocks) which increase pair formation with the target RNA or target DNA are preferably arranged in the more central sequences of the oligonucleotide, for example between in each case the fourth last, or in each case the third last, or in each case the second last, or in each case the last, building blocks of the sequence. In an oligonucleotide having, for example, 20 building blocks, pairing building blocks are preferably located in the region from the fourth to the seventeenth building block.
In a particular embodiment, R3, as a biological active group, is preferably a sugar molecule or its derivative; more preferably, R3 is a sugar molecule residue of a glycolipid, glycoprotein or polysaccharide; particularly preferably, R3 is N-acetylglucosamine or its derivatives, glucose or its derivatives, lactose or its derivatives, sialyl-Lewis x or its derivatives, or sialyl-Lewis a. As a biological active group, R3 is preferably biotin.
The invention additionally relates to a process for preparing polypeptides from at least one structural element of the formula V or at least two different structural elements of the formula (V) and, if desired, structural elements of the formulae I, II and IV, 
in which A, R1, X1, R2, X2 and R3 are as defined above, which comprises reacting polypeptides having structural elements of the formula I, and, if desired, structural elements of the formulae II and/or IV, 
in which A, R1, X1, R2, X2 and X3 are as defined above, with a thiol of the formula VIII,
R3xe2x80x94SHxe2x80x83xe2x80x83(VIII)
in which R3 is as defined above, in the presence of a strong, non-nucleophilic base having at least one tertiary N atom.
The thiols are either known or can be prepared by methods which are known per se. It is particularly advantageous to introduce the thiol group by reacting compounds of the formula R3YH with thiolactones, with xe2x80x94YH being a functional group, for example xe2x80x94OH or NH2.
Suitable and preferred bases are bicyclic or polycyclic amines having at least one tertiary N atom. Examples are quinuclidine and 1,8-diazabicyclo[5.4.0]undec-7-ene(1,5-5) (DBU).
The base is employed in at least equimolar quantities, preferably in slight excesses.
It has been found, surprisingly, that it is possible to achieve quantitative reactions and high purities so that the polypeptides can be used directly without further elaborate purifications, even in the biological sphere. The reaction can also be carried out using alkali metal thiolates R3SM (M is alkali metal)
The reaction can be effected in the presence of a polar, aprotic solvent. Those which are preferred are nitrogen-dialkyiated carboxyimides and lactams, sulfoxides and sulfones, for example dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide and tetramethylene sulfone.
The reaction can be effected either with or without the addition of water, for example up to quantities at which the polymer remains in solution.
The temperature range in which the reaction can be effected extends, for example, from 0 to 200xc2x0 C., preferably from 0 to 150xc2x0 C., particularly preferably from 20 to 100xc2x0 C. and in particular from 20 to 50xc2x0 C.
The invention furthermore relates to the novel compounds for use in a therapeutic method for treating diseases in homoiothermic animals, including man. The dosage when administering to homoiotherms of about 70 kg bodyweight can, for example, be from 0.01 to 1000 mg per day. Administration is preferably effected parenterally, for example intravenously or intraperitoneally, in the form of pharmaceutical preparations.
The compounds according to the invention have antiinflammatory properties and can accordingly be used as medicaments. It is possible with them in particular to alleviate disorders such as cardiogenic shock, myocardial infarct, thrombosis, rheumatism, psoriasis, dermatitis, acute respiratory distress syndrome, asthma, arthritis and metastatic cancer.
The invention furthermore relates to a pharmaceutical preparation which comprises an effective quantity of the novel compound, either alone or together with other active ingredients, a pharmaceutical carrier, preferably in a significant quantity, and adjuncts, if desired.
The pharmacologically active, novel compounds can be used in the form of parenterally administerable preparations or infusion solutions. These solutions are preferably isotonic, aqueous solutions or suspensions, with it being possible, for example in the case of lyophilized preparations, which comprise the active substance either alone or together with a carrier, for example mannitol, to prepare the latter prior to use. The pharmaceutical preparations can be sterilized and/or comprise adjuncts, for example preservatives, stabilizers, wetting agents, emulsifiers, solubilizers, salts for regulating the osmotic pressure and/or buffers. The pharmaceutical preparations, which, if desired, can also comprise additional pharmacologically active substances, for example antibiotics, are prepared in a manner known per se, for example using conventional solubilizing or lyophilizing methods, and comprise from about 0.1% to 90%, in particular from about 0.5% to about 30%, for example from 1% to 5%, of active ingredient(s).
The polypeptid polymer according to the invention can be used to detect diseases in homeothermic animals including man.
The invention is further directed to a method of using of a polypeptid polymer according to the invention for production of monoclonal antibodies.
The polypeptid polymer according to the invention can be used in a ligand binding assay, preferably for determining the concentration of a compound required to inhibit maximal binding of the polypeptide polymer according to the invention to an immobilized receptor molecule. The immobilized receptor molecule can, according to example D1 of the specification, be an immobilized E-selectin/human lgG chimera or can according to example D2 be an immobilized P-selectin/human IgG chimera.
The linear polymers, which present ligands in a multivalent form, may be employed in the medical sphere as pharmaceuticals for therapy, both in animals and in human medicine.
The effect of the pharmaceuticals is amplified by the multivalent presentation. Alkaloids, vitamins, hormones, antibiotics, toxins and cytostatic agents, for example, can be administered as pharmaceuticals, bound to the linear polymers. The antigens which are bound to the linear polymers can be used as vaccines and can be employed as conjugates with the polymer for active immunization in animals or humans. The conjugates may likewise be employed for preparing monoclonal antibodies against the given hapten structures.
The linear polymers which present ligands in multivalent form can be used, as water-soluble, synthetic polymers, as an artificial replacement for plasma.
Ligands which are selectively recognized by particular receptors can be bound to the linear polymers and selectively direct pharmaceuticals, which are also present, to their sites of action (drug targeting).
The polypeptide according to the invention wherein the biological active group R3 is a ligand which is selectively recognized by a particular receptor can be used to target molecules to their side of action by attaching said molecules to the polypeptide polymer and binding the so modified polypeptide polymer to its particular receptor such that the receptor and the target molecule are in close proximity. The molecules which are targeted can be selected from a group consisting of drugs, genes, biological active proteins and fluorescent polymer coated beads.
The polypeptide according to the invention can be used to select cells, wherein the biological active group R3 is a ligand for proteins or receptors expressed on the selected cell type.
The polypeptide according to the invention can be used in a method for selecting cells by using a polypeptide according to the invention, wherein the biological group R3 is a ligand for proteins or receptors expressed on the selected cell type. The method for selecting cells can be selected from a group consisting of panning on immobilized polypeptides, cell chromatographie or fluorescence activated cell sorting.
The polypeptide according to the invention can be used to inhibit binding of cells type A to cells of type B, wherein the biological active group R3 is an inhibitor which upon incubation of cells of type A or upon incubation of cells of type B or incubation of both prevents binding of cells of type A with specific ligands bound to their surface to a particular receptor expressed on cells of type B.
The polypeptide according to the invention can preferably be used to inhibit binding of polymorphonuclear leukocytes (PMNs) with Sialyl Lewis x groups bound to their surface, to Human Umbilical Vein Endothelial cells (HUVECS) which upon stimulation with Tumor Necrosis Factor a (TNF) express E-selectin on their surface.
The polypeptide according to the invention can be used to study the rolling behavior of cells of type A on the confluent layer of cells of type B, which is a characteristic of cells of type A in contact with a receptor expressed on cells of type B in presence of hydrodynamic flow. According to Example D3 the cells of type A are preferably polymorphonuclear leukocytes (PMNs) with Sialyl Lewis x groups bound to their surface, and cells of type B are preferably Human Umbilical Vein Endothelial cells (HUVECS) which upon stimulation with Tumor Necrosis Factor a (TNF) express E-selectin on their surface
The present invention is further directed to a composition for determining the concentration of a compound required to inhibit maximal binding of a polypeptide polymer according to the invention to an immobilized receptor molecule in form of a ready-to-use test kit, comprising in addition to the carrier materials, reagents and other additives customarily used at least one polypeptide polymer according to the invention. The ready-to-use test kit preferably comprises a carrier which is compartementalized so as to receive, in close mutual confinement therein:
a) a solid support having affixed thereto the receptor molecule capable of reacting with the polypeptide polymer according to claim 25 and
b) a detecting system for a binary complex. The present invention is preferably directed to a composition used according to Examples D1 and D2.
The present invention is further directed to a composition for quantification of the rolling behavior of the cells of type A in the presence of a hydrodynamic flow on the confluent layer of cells B in form of a ready-to-use test kit, comprising in addition to the carrier materials, reagents and other additives customarily used at least one polypeptide polymer according to the invention.
The polypeptid polymer according to the invention can be used for immunization in animals or humans, wherein the antigens are bound to the linear polymers.
Under defined physiological conditions, pharmaceuticals which are bound to the linear polymers by way of labile bonds can either be released over a very long period (slow release) or at specific sites of action (controlled release). This is particularly effective in the case of combination preparations.
Polymeric compounds which present ligands in multivalent form may be employed as receptor blockers, for example, in ligand-receptor recognition processes.
The present invention is preferably directed to a composition used according to Examples D3.
The ligands which are bound to the linear polymers are presented in multivalent form by the recurring structural elements of the polymer. These polymers are particularly valuable when used as the recognition component in an analytical test system.
In the sphere of medical diagnosis, the linear polymers which present ligands in multivalent form can be used in an immunoassay for the early recognition of diseases by detecting specific indicator substances, for example in the diagnosis of cancer or for diagnosing viral or bacterial diseases or for diagnosing parasite infestation. In this context, the recognition component can be immobilized antibodies for detecting specific antigens or immobilized antigens for detecting specific antibodies. The immobilized antigen can, for example, be a surface protein of the HIV virus and consequently be used for diagnosing HIV infections.
In the field of analytical environmental diagnosis, immobilized antibodies can be employed, in an immunoassay, as a probe for antigens and consequently be used, for example, for detecting plant protectants, such as atrazine.
In the field of medical diagnosis, immobilized oligonucieotides can be employed, in a test system, as a probe for target oligonucleotide sequences, for genomic analysis, or for diagnosing genetically determined diseases, such as cystic fibrosis.
In sensor technology, the linear polymers which present ligands in multivalent form can be included, as recognition elements, in the active layer, both in optical and in electrochemical sensors, with it being possible for the recognition element to be an antibody, an antigen or an oligonucleotide which is immobilized on the sensor surface. Examples of the recognition element correspond to the abovementioned examples in the fields of medical or environmental diagnosis.
Insecticides which are bound to the linear polymers can be employed for controlling insects.
The following examples clarify the invention. In the examples which follow, the following abbreviations are used:
The {overscore (M)}w of the polylysine hydrobromides which are commercially obtainable from SIGMA were determined by means of viscosity measurements and SEC-LALLS (size exclusion chromatography/low angle laser light scattering) of the succinyl derivatives of the compounds.